Inhibition of DUX4 Expression Using Bromodomain and Extra-Terminal Domain Protein Inhibitors (BETi)

ABSTRACT

The use of BETi as a potential treatment for FSHD is provided. Specifically, the use of BETi, and particularly selective BETi for BRD4, are shown to inhibit DUX4 expression which is expected to result in a decrease in the severity of symptoms of FSHD. Further, the treatments are shown to work when pulsed as opposed to continuous. This allows for a BETi to be supplied to a human in a pulse, and then allows the human to not need any additional treatment for a window at least as long as the one of the treatment pulse.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a Continuation of U.S. Utility patent applicationSer. No. 14/491,599 filed Sep. 19, 2014 and currently pending which, inturn, claims benefit of U.S. Provisional Patent Application Ser. No.61/880,565, filed Sep. 20, 2013 and now expired. The entire disclosureof all the above documents is herein incorporated by reference.

BACKGROUND 1. Field of the Invention

Discussed herein are systems and methods for the inhibition ofbromodomain and extra-terminal domain (BET) proteins to block inductionof DUX4 mRNA and protein expression activity and assist in treatment offacioscapulohumeral muscular dystrophy (FSHD).

2. Description of the Related Art

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most commontype of muscular dystrophy (akin in prevalence to Duchenne musculardystrophy and myotonic dystrophy) affecting approximately 12 in 100,000.It is a genetic muscle disorder that initially affects the muscles ofthe face, shoulder blades and upper arms. However, muscle degenerationis progressive and spreads to other muscles including those in the legsand hips. The disorder is most commonly detected due to muscle weaknessin the shoulder blades resulting in muscles that no longer serve tocorrectly support the shoulder blades as a fulcrum for the arm. Thus,the shoulder blades often have excessive movement which can result indifficulty throwing or raising the aims above the head. Weakness offacial muscles resulting in difficulty puckering (and related activitiessuch as drinking through a straw or whistling) are also common.

FSHD usually begins prior to age 20 and the disorder is progressive withmuscle degeneration continuing throughout life. Sometimes FSHD isdivided between adult-onset and infantile-onset forms with the adultonset (which includes onset in adolescence) being more common. Becauseit is characterized by progressive muscle weakness, in some individualsmuscle weakness will eventually spread to the lower extremities, oftenresulting in affected individuals becoming wheelchair-bound. Further,pain and fatigue are frequent complaints and are presumably associatedwith muscle degeneration.

FSHD is almost always associated with a genetic flaw (mutation) thatleads to a shorter than usual segment of DNA on chromosome 4.Specifically, FSHD type 1 (FSHD1) accounts for 95% of FSHD cases and isassociated with contraction of the number of D4Z4 repeat units in themacrosatellite array at 4q35. FSHD2 (˜5% of cases) occurs in the absenceof D4Z4 contraction and is believed caused by haplo-insufficiency ormutation of the SMCHD1 gene. Symptoms and effects of both types aregenerally the same.

The polymorphic D4Z4 array varies between 11 and >100 copies in healthyindividuals, while FSHD1 patients retain 1-10 repeat units. Each repeatunit contains a copy of the DUX4 retrogene. While the disorder isbelieved to be genetically passed on, it also may occur spontaneously incertain cases.

There is currently no treatment available for FSHD and no clinicaltrials of promising treatments are ongoing. Nonsteroidalanti-inflammatories, or NSAIDs, are sometimes prescribed to providecomfort and mobility, but they have no effect on the underlying disease.Similarly, orthoses are often used to help support parts of the bodywhere muscle degeneration is resulting in insufficient support, andcertain types of surgery such as thoracoscapular fusion, where theshoulder blade is fused to the ribs to provide for more rigid supportfor the arms, are used in similar fashion. While these options can oftenincrease mobility and function, they also have no effect on theunderlying degeneration and therefore serve only to provide for comfort,as opposed to cure.

Despite recent advances in our understanding of the epigenetic mechanismof FSHD pathology, little insight has been gained into specifictherapeutic targets amenable to small molecule drug intervention. Drugintervention can be beneficial for disorders such as FSHD as it can besignificantly less invasive than surgery and, should it be able totarget an underlying cause of the degeneration, can potentially furtherslow, halt, or possibly reverse the degeneration itself. Druggabletargets that regulate DUX4 expression and evaluation of the therapeuticpotential of the corresponding inhibitory compounds are desirable.

SUMMARY

The following is a summary of the invention in order to provide a basicunderstanding of some aspects of the invention. This summary is notintended to identify key or critical elements of the invention or todelineate the scope of the invention. The sole purpose of this sectionis to present some concepts of the invention in a simplified form as aprelude to the more detailed description that is presented later.

The use of BET bromodomain protein inhibitors (BETi) as a potentialtreatment for FSHD is provided. Specifically, the use of BETi, andparticularly selective BETi for BRD4, are shown to inhibit DUX4expression which is expected to result in a decrease in the severity ofsymptoms of FSHD. Further, the treatments are shown to work when pulsedas opposed to continuous. This allows for a BETi to be supplied to ahuman in a pulse, and then allows the human to not need any additionaltreatment for a window at least as long as the one of the treatmentpulse.

There is described herein, among other things, a method of reducingmuscular weakness from facioscapulohumeral muscular dystrophy (FSHD),the method comprising: administering to a patient with FHSD atherapeutic quantity of a BET inhibitor (BETi) sufficient to reduce DUX4expression in said patient.

In an embodiment of the method, the BETi is selected from the groupconsisting of: (+)-JQ1, PFI-1, I-BET-762, I-BET-151, RVX-208, andCPI-0610.

In an embodiment of the method, the BETi is a selective inhibitor ofBRD2.

In an embodiment of the method, the BETi is a selective inhibitor ofBRD4.

In an embodiment of the method, the BETi is a broad spectrum inhibitor.

In an embodiment of the method, in said administering, said BETi isprovided in an initial dose and a second dose is not provided for atleast 24 hours after said initial dose is finished.

In an embodiment of the method, said initial dose is providedcontinuously over 24 hours.

In an embodiment of the method, in said administering, said BETi isprovided in an initial dose and a second dose is not provided for atleast 48 hours after said initial dose is finished.

In an embodiment of the method, in said administering, said BETi isprovided in an initial dose and a second dose is not provided for atleast 72 hours after said initial dose is finished.

In an embodiment of the method, in said administering, said BETi isprovided in an initial dose and a second dose is not provided for atleast 96 hours after said initial dose is finished.

In an embodiment of the method, the patient is human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph indicating that DUX4 expression strongly inducesthe ZSCAN4 promoter. A luciferase reporter plasmid containing thepromoter and enhancer from the ZSCAN4 gene was transfected into normalmyoblasts in the absence or presence of a DUX4 expression plasmid.Results are expressed as fold increase in luciferase activity normalizedto the activity of the ZSCAN4 reporter in the absence of exogenous DUX4expression with error bars indicating standard deviations.

FIG. 2 shows a graph indicating that the ZSCAN4 promoter is induced bymyoblast differentiation only in FSHD (54-2) but not normal (54-6)myotubes. In addition, ZSCAN4 promoter induction requires the presenceof DUX4 binding sites since mutation of these sites (ZSCAN4.mut) blockspromoter induction. Results are expressed as fold increase in luciferaseactivity normalized to the activity of the ZSCAN4 reporter inundifferentiated FSHD myoblasts.

FIGS. 3A and 3B show graphs indicating that DUX4 and ZSCAN4 mRNA levelsare induced during FSHD1 myoblast differentiation. Results are expressedas relative expression normalized to levels in undifferentiated cellswhich were set to one.

FIG. 4 shows a graph of the inhibition of ZSCAN4 promoter activity indifferentiated FSHD myoblasts by BETi (+)-JQ1 and PFI-1. Results areexpressed as relative luciferase units (RLUs) with IC₅₀s indicated. Notethat concentration is plotted on a log scale.

FIG. 5 shows a graph of ZSCAN4 mRNA levels determined by qRT-PCR inundifferentiated FSHD1 myoblasts, 6 day differentiated myoblasts anddifferentiated myoblasts treated with (+)-JQ1. Error bars indicatestandard deviations.

FIG. 6 shows a graph indicating that (+)-JQ1 inhibits the induction ofDUX4 mRNA by differentiation. Error bars indicate standard deviations.

FIG. 7 shows a graph indicating that DUX4 activity is not blocked by(+)-JQ1. A DUX4 expression plasmid was co-transfected with the ZSCAN4reporter plasmid and cells immediately treated with compound. Resultsare normalized to activity in the absence of exogenously expressed DUX4.

FIGS. 8A and 8B show graphs of the levels of mRNA for ZSCAN4 and TRIM43(downstream targets for DUX4) in undifferentiated myoblasts. FIG. 8Ashows the level of ZSCAN4, and FIG. 8B shows the level of TRIM43. FSHD1(54-2) or normal (54-6) myoblasts were treated with DMSO (control) orcompounds (+)-JQ1 (500 nM) or PFI-1 (2 μM) as indicated for 48 hoursbefore RNA isolation. mRNA levels are normalized to the FSHD1 cells DMSOcontrol using RPL13A as the internal control.

FIGS. 9A and 9B show graphs of the levels of ZSCAN4 mRNA after treatmentof subconfluent FSHD1 myoblasts with BETi. FIG. 9A shows the ZSCAN4 mRNAlevels after treatment with (+)-JQI for 24, 48, and 72 hours. FIG. 9Bshows the ZSCAN4 mRNA levels after treatment with BETi (+)-JQ1, I-BET762and I-BET151 for 72 hours at the indicated concentrations.

FIGS. 10A and 10B show graphs of the levels of ZSCAN4 mRNA after growthof FSHD myoblasts in the presence of low concentration (+)-JQ1 (100 nM)and during a time course after withdrawal of (+)-JQ1. FIG. 10A showsresults using FSHD1 myoblasts. FIG. 10B shows results using FSHD2myoblasts.

FIGS. 11A, 11B, and 11C show siRNA knockdown of BET family members inFSHD myoblasts. FSHD1 myoblasts were transfected with the indicatedsiRNAs and RNA analyzed 96 h later by qRT-PCR.

FIG. 12 shows BET family protein levels after siRNA knockdown. Cellswere transfected as above and 72 h later cell lysates were analyzed bywestern blot with the indicated antibodies.

FIGS. 13A, 13B, and 13C shows DUX4 target gene expression after siRNAknockdown. Cells were transfected as above and RNA analyzed 96 h laterby qRT-PCR. Error bars indicate standard deviations.

FIGS. 14A, 14B, 14C, 14D, 14E, 14F, and 14G show the exposure-responserelationship of DUX4 target and non-target gene expression in FSHD1myoblasts treated with I-BET762 for various length of time. FIG. 14Ashows the timeline. Subconfluent myoblasts were treated I-BET762 (Cmpd)for either 8, 24, 48 or 72 hours and gene expression analyzed at the 72hour time point. FIGS. 14B, 14D, and 14F show DUX4 target geneexpression determined by qRT-PCR. FIGS. 14C, 14E, and 14G shown non-DUX4target gene expression.

FIGS. 15A, 15B, and 15C show the duration of DUX4 target gene expressionresponse following a 24 hour treatment of FSHD myoblasts with I-BET762.FIG. 15A shows the experimental timeline. FSHD1 myoblasts were treatedwith a range of concentrations of I-BET762 for 24 hours. Compound wasthen removed, the cells rinsed once with growth media and fresh mediare-applied to cells. RNA was harvested at days 3, 4, 6, and 8 asindicated for analysis by qRT-PCR. FIG. 15B shows ZSCAN4 RNA levelsversus I-BET762 concentration and FIG. 15C shows MBD3L2 RNA levelsversus I-BET762 concentration. EC₅₀ curves were plotted at each timepoint.

FIGS. 16A and 16B shows a 24 hour exposure of differentiating FSHDmyotubes to (+)-JQ1 sustains a decrease in DUX4 target gene expressionwith minimal effect on differentiation. (+)-JQ1 (500 nM) was added onday 3 (D3) of differentiation and removed on D4. RNA was isolated forqRT-PCR analysis at the indicated time points. FIG. 16A shows ZSCAN4 RNAlevels and FIG. 16B shows Myosin heavy chain (MYH2) RNA levels.

FIGS. 17A and 17B show continuous exposure of FSHD myotubes to RVX-208during 72 h of differentiation blocks the induction of the DUX4 targetZSCAN4 without affecting the late differentiation marker myosin heavychain (MHY2).

DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

Discussed herein is the use of a cell based assay to detect DUX4expression in FSHD muscle cells. The connection of DUX4 expression toFSHD is used to screen a library of compounds that target epigeneticmodifier proteins to locate a potential treatment for FSHD. This“chemical genetics” approach identified the bromodomain and extraterminal (BET) family of proteins as key targets involved in DUX4expression. Selective inhibitors of BET proteins (BETi), specifically(+)-JQ1, PFI-1, RVX-208 (Resverlogix Corp.), I-BET-762(GlaxoSmithKline), and I-BET-151 (GlaxoSmithKline) were found to blockthe induction of DUX4 and its downstream targets ZSCAN4 and TRIM43during differentiation of FSHD1 and FSHD2 muscle cells in vitro.

Further, BETi also block DUX4 expression in undifferentiated FSHDmyoblasts. While this disclosure will focus on the use of JQ1 andcertain other BETi, it should be recognized that specific BETi can beused in a variety of different situations and a large number ofpotential BETi are known. This includes using BETi such as, but notlimited to, those described in BET bromodomain inhibitors: a patentreview Gamier et al. (informa healthcare (2014)) the entire disclosureof which is herein incorporated by reference.

Also, ZSCAN4 and TRIM43 mRNA levels were analyzed 48 to 72 hours afterthe BETi was added. Cells grown continuously in a low dose BETi JQ1exhibited a greater than 95% reduction in steady state levels of ZSCAN4mRNA and this effect was sustained, requiring longer than 7 days torecover after removal of JQ1 from the cultures. This data suggests thatBETi generally and (+)-JQ1, PFI-1, I-BET-762 (GlaxoSmithKline), andI-BET-151 (GlaxoSmithKline) specifically, have therapeutic value inFSHD. RVX-208 (Resverlogix Corp.) and CPI-0610 (ConstellationPharmaceuticals) as BETi are also expected to have therapeutic value inFSHD.

Muscle pathology in both FSHD1 and FSHD2 coincides with the epigeneticde-repression of the normally silent DUX4 gene. In healthy individuals,DUX4 is expressed early in development but then becomes silenced insomatic cells as part of sub-telomeric heterochromatin. DUX4 is adouble-homeobox transcription factor and its abnormal expression in FSHDindividuals initiates a transcriptional program that results in musclecell dysfunction and death. Although the genetic defects differ in FSHD1and FSHD2, the clinically indistinguishable outcomes indicate aconvergence in pathways responsible for DUX4 expression. Chromatin isde-condensed at 4q35 in muscle cells from both FSHD1 and FSHD2 patients;however, DUX4 expression is variegated and an extremely rare event invivo and in vitro. Less than 1 in 1000 FSHD myoblasts in culture expressDUX4 in stochastic “bursts” of expression. The proportion of nucleiexhibiting bursts of DUX4 expression increases upon myotubedifferentiation.

It is believed that a number of events subsequent to general chromatinde-condensation occur to fully de-repress DUX4. Therefore, knowledge ofthe epigenetic mechanisms behind the bursts of DUX4 expression allowsfor the development of therapeutic strategies to maintain the repressedstate of the DUX4 gene. Specifically, the use of inhibitors of BETproteins (BETi) would appear to provide therapeutic effects to thosewith FSHD by silencing of DUX4 expression.

Recent work has identified Polycomb group (PcG) repression as the keymechanism governing the silencing of DUX4 and has implicated Trithoraxgroup (TrxG) mechanisms in de-repression. PcG and TrxG proteins areevolutionarily-conserved complexes originally identified in Drosophilathat regulate chromatin structure and are required to maintainestablished gene expression patterns (i.e. epigenetic memory). PcGproteins comprise two major enzymatic complexes whose activitiescontribute to chromatin compaction: Polycomb repressive complex 1 (PRC1)and PRC2. PRC1 contains an E3 ubiquitin ligase and catalyzesubiquitination of lysine 119 on histone H2A. PRC2 catalyzes the di- andtri-methylation of lysine 27 on histone H3 (H31(27 me2/me3). TrxGproteins encompass a variety of biochemical activities includingchromatin remodeling, histone modification, and transcription factor(trans-activating) functions that antagonize PcG proteins to de-repressPcG targets. How TrxG proteins counteract PcG repression is not fullyunderstood, but has been recognized. In Drosophila, two histonemethyltransferases that are part of TrxG complexes, absent, small orhomeotic discs 1 (ASH1) and trithorax (TRX) are thought to counteractPcG silencing through histone methylation activity, though some havechallenged this simple view.

PcG and TrxG bind to specific DNA regions termed Polycomb/Trithoraxresponse elements (PREs/TREs) in Drosophila. Each human D4Z4 repeatcontains multiple elements that correspond to Drosophila PREs, andPcG-mediated repression of 4q35 genes appears to depend on D4Z4 copynumber. Recently, new insights into how TrxG proteins are involved inovercoming PcG repression to de-repress DUX4 gene expression have beenuncovered. ASH1L, the mammalian homolog of fly ASH1, was shown to berecruited to D4Z4 specifically in FSHD myoblasts by the D4Z4-encodedlong noncoding RNA (ncRNA) DBE-T. ASH recruitment was associated with anincrease in H3K36me2 and DUX4 de-repression. However, the full mechanismby which DBE-T and ASH1L functions to de-repress DUX remains to beelucidated.

Epigenetic modifications controlling chromatin compaction and access toregulatory factors include DNA methylation and post-translationalmodifications of histones including methylation, acetylation,ubiquitination and phosphorylation. Additionally, a number of non-codingRNAs, such as DBE-T described above, play a role as epigeneticmodifiers. FSHD is considered an “epigenetic disease” due to the factthat chromatin at D4Z4 is de-condensed and exhibits correspondinglyaltered epigenetic marks including loss of DNA methylation, decreasedH3K9me3 with decreased heterochromatin protein 1 gamma and cohesinbinding, and decreased PcG silencing mark H3K27me3 specifically atcontracted D4Z4 alleles. These epigenetic modifications are processed bya large number of “epigenetic modifiers” that are broadly classified bytheir functions as “writers,” “readers” and “erasers” that,respectively, add, detect by binding, or remove various chemicalmodifications.

The specific marks altered in disease states such as FSHD provideinformation as to the epigenetic machinery that is most closely involvedwith the disease process. For example, decreased enrichment for theH3K27me3 mark implicates a defect in PcG silencing while increasedH3K36me2 suggests TrxG activation. Another approach to gain informationabout disease states is to screen compounds that inhibit defined targets(chemical genetic probes) against disease-relevant biological assays.Recent advances towards epigenetic interventions for cancer and otherdiseases have resulted in the development of many novel chemical probes.These include marketed drugs, compounds in clinical trials and manyother compounds that target specific proteins whose signals contributeto epigenetic gene regulation. The availability of chemical modulatorsof epigenetic targets provides a unique opportunity to identify novelpathways relevant to FSHD. Applying these reagents in a “chemicalgenetics” approach helps define the mechanism of aberrant generegulation in FSHD and lead to therapeutic interventions that addressthe underlying epigenetic mechanism of pathology.

The germline transcription factor DUX4 induces a complex pattern ofinappropriate gene expression in FSHD muscle, including activation ofearly stem cell and germline programs. A complicating factor is thatDUX4 can only be detected in approximately 1 in 1000 myoblasts in vitro,making DUX4 mRNA difficult to detect. However, the downstream targets ofDUX4, most of which are undetectable in normal myoblasts, are readilydetectable in FSHD myoblasts. One of these DUX4 target genes, ZSCAN4,contains 4 tandem DUX4 binding sites within its enhancer/promoterregion. A luciferase reporter vector containing the enhancer/promoterregion from ZSCAN4 can serve as a sensitive indicator of DUX4 expressionand, indirectly, the chromatin state at the DUX4 locus. FIG. 1 shows aca. 3000-fold induction of the ZSCAN4 luciferase reporter inco-transfection experiments with a DUX4 expression plasmid in myoblasts.These data indicate that the ZSCAN4 luciferase reporter is highlysensitive to exogenously expressed DUX4.

To test responsiveness to endogenously expressed DUX4, the wt ZSCAN4reporter or a mutant reporter in which 3 of the 4 DUX4 binding siteswere mutated and were transfected into FSHD1 (54-2) and control (54-6)myoblasts derived from a mosaic patient. The wt ZSCAN4 luciferasereporter elicited only weak and sporadic activity in undifferentiatedFSHD myoblasts (not shown). Based on observations that DUX4 is inducedwhen myoblasts differentiate to myotubes, undifferentiated cells tothose induced to differentiate for 6 days were compared. FIG. 2 showsthat ZSCAN4 promoter activity is induced about 10-fold upondifferentiation of FSHD1 but not control myoblasts. In addition, thisinduction requires intact DUX4 binding sites since the mutant reporterwas not equally induced. Similar results are obtained when using FSHD2cells (not shown). Together, these data suggest that ZSCAN4 reporterreadout represents the de-repression of DUX4 expression that occurs onlyin the context of an FSHD genetic background.

To confirm that the increase in ZSCAN4 luciferase reporter readoutmirrored an increase in DUX4 and ZSCAN4 expression, mRNA from duplicatesamples was isolated and analyzed by qPCR. As mentioned above, DUX4message is expressed only in a small subset of cells and is difficult todetect. In addition, two forms of the mRNA are present, a non-proteincoding short Rhin that is present in both normal and FSHD muscle cells(DUX4s) and a DUX4-encoding full length form (DUX4fl). A TaqMan-styleqPCR assay to specifically detect DUX4fl was not available and an assaydetecting both DUX4s and DUX4fl was used for detecting the increase inDUX4fl that accompanies FSHD myotube differentiation. FIGS. 3A and 3Bshow that under the conditions of the screening assay, DUX4 mRNA levelsare induced nearly 90-fold upon myoblast differentiation and ZSCAN4 mRNAlevels are induced greater than 200-fold. These data indicate that thescreening assay does report bona fide DUX4 de-repression and associatedZSCAN4 induction.

Based on the above, differentiation of FSHD myoblasts and a concomitantincrease in ZSCAN4 promoter activity represent a model of DUX4de-repression that is amenable to screening compounds. Two smalllibraries of compounds targeting known epigenetic modifying enzymes wereobtained: Cayman Chemicals “Epigenetic Screening Library” and SelleckChemicals “Epigenetic Compound Library”. Together they represent about100 modulators of known epigenetic “writer”, “reader” and “eraser”proteins. These compounds were screened for their activity in modulatingthe induction of ZSCAN4 promoter activity in differentiating FSHDmyoblasts. A co-transfected internal control plasmid that expressedRenilla luciferase was used to monitor general compound toxicities aswell as non-specific transcriptional effects. There were six categoriesof “hits” identified as blocking the induction of Firefly luciferase(ZSCAN4 reporter) without significantly inhibiting Renilla luciferaseactivity (control).

Notably, materials generally identified as BET bromodomain inhibitors(BETi) (2 separate hits) resulted in nearly complete inhibition ofZSCAN4 promoter induction and would likely treat or help eliminatesymptoms of FSHD. Inhibition curves for the BET bromodomain inhibitor(+)-JQ1 and PFI-1 are shown in FIG. 4. The IC₅₀s are consistent withreported cellular assay potencies for each compound. Additionally, theseinhibitors were active in both FSHD1 and FSHD2 myoblasts, suggestingthat they act at a point in the epigenetic switch controlling DUX4expression common to both genetic defects.

The reporter readout in the screening assay is designed to reflect theendogenous response of the ZSCAN4 promoter. Thus, an important secondaryassay is to confirm that screening hits block the induction ofendogenous ZSCAN4 mRNA. TaqMan qRT-PCR was used to measure ZSCAN4 mRNAlevels after compound treatment of differentiating FSHD1 myoblasts. Theendogenous control mRNA chosen for normalization was the ribosomalprotein L13A (RPL13A). FIG. 5 shows that the BETi (+)-JQ1 blocksinduction of ZSCAN4 mRNA upon differentiation of FSHD myoblasts. Similarresults were seen for each compound when using FSHD2 myoblasts.Additionally, the levels for another DUX4-induced gene, TRIM43 weremeasured. Similar to ZSCAN4, TRIM43 mRNA was induced by differentiationof FSHD myoblasts and its induction blocked by (+)-JQ1 (not shown),providing evidence that this compound blocks multiple downstream targetsof DUX4 and therefore is suitable for use as a therapy for FSHD.

While not intending to be limited by any theory of operation, blockinginduction of ZSCAN4 mRNA may occur by one of several mechanisms: i)blocking de-repression of DUX4, ii) interfering with DUX4 activitysubsequent to its de-repression and iii) via a DUX4-independentmechanism. Levels of DUX4 mRNA by qRT-PCR in the samples from compoundtreatment were measured. FIG. 6 demonstrates that (+)-JQ1 blocks theinduction (de-repression) of DUX4 mRNA that accompanies differentiationof FSHD1 myoblasts. Similar results are seen when using FSHD2 myoblast(not shown). To ensure that (+)-JQ1 did not interfere with DUX4activity, a co-transfection experiment was performed in which the ZSCAN4luciferase reporter construct was transfected into normal myoblastsalong with a DUX4 expression plasmid similar to the experiment inFIG. 1. Three hours after transfection was started, (+)-JQ1 was added atconcentrations up to twenty fold higher than needed to completely blockDUX4 de-repression in the differentiation model. Cells were harvestedfor luciferase activity 24 hours later. FIG. 7 demonstrates that even ata 10 μM concentration, (+)-JQ1 had only a marginal (not statisticallysignificant) effect on DUX4 activity. These data further support that(+)-JQ1 inhibits the expression of DUX4 rather than its activity.

Based on the above, there is an about 90-fold induction of DUX4 mRNAupon differentiation of FSHD myoblast for 6 days. The compound (+)-JQ1appears to block this induction. Since the induction of DUX4 coincideswith muscle cell differentiation, it is possible that (+)-JQ1 actsindirectly by blocking muscle cell differentiation. (+)-JQ1 affectedmarkers of muscle cell differentiation, however, particularly inhibitingthe appearance of the late differentiation marker myosin heavy chain(MYH2).

By testing the effects of (+)-JQ1 in undifferentiated myoblasts, it ispossible to avoid the complication of inhibiting the muscledifferentiation program. Undifferentiated FSHD1 myoblasts were treatedwith the BETi (+)-JQ1 or PFI-1 for 24 and 48 hours and a qRT-PCR wasperformed to determine mRNA levels for DUX4, ZSCAN4 and TRIM43. As notedbefore, the TaqMan primer/probe set detects both DUX4s (expressed innormal and FSHD myoblasts, non-protein encoding) and DUX4fl (expressedonly in FSHD myoblasts, DUX4-encoding). Since DUX4fl is expressed athard to detect levels in undifferentiated FSHD myoblasts, qPCR signalsrepresenting the short DUX4 message, or D4Z4 transcripts from othergenomic regions present at very low levels interfere with the signal forDUX4fl and make direct qPCR difficult.

Therefore, a more sensitive and accurate indicator of DUX4 expression inundifferentiated FSHD myoblasts is the amplified signal of itsdownstream targets ZSCAN4 and TRIM43. FIGS. 8A, and 8B show that thelevels of ZSCAN4 and TRIM43 are significantly decreased by 48 hourtreatment with both (+)-JQ1 and PFI-1, suggesting that blocking theexpression of DUX4 with BETi is independent of muscle celldifferentiation. Interestingly, the kinetics of the response appears tobe slow since there was no decrease with 24 hours of compound treatmentdetected (FIG. 9A). While not intending to be bound by any theory ofoperation, it is hypothesized that BETi blocks the de-repression of DUX4that occurs stochastically at a very low frequency in undifferentiatedmyoblasts. The expression of downstream targets is sustained such thatdecreases in their mRNA levels require blocking new DUX4 expression overthe course of a longer time frame (e.g. 48 hours or more). Theexperiment depicted in FIGS. 8A, 8B, and 8C was performed insemi-confluent monolayers of FSHD1 myoblasts. These results demonstratedthat two BETi (PFI-1 and (+)-JQ1) reduce the expression of DUX4downstream targets ZSCAN4 and TRIM43 after 48 hours of treatment,excluding the possibility that inhibition of DUX4 expression isdependent on differentiation. Longer term exposure to (+)-JQ1 results ina more dramatic decrease in the expression of downstream targets (asshown in FIGS. 9A-10B).

FIG. 9A shows a time course of ZSCAN4 mRNA levels during treatment ofsubconfluent FSHD1 myoblasts with (+)-JQ1. Under these conditions,dramatic (>95%) decreases in ZSCAN4 mRNA are evident at both 48 hoursand 72 hours of compound treatment suggesting that effects of BETi onZSCAN4 mRNA occur faster in dividing cells. FIG. 9B shows the effects ofadditional BETi (I-BET762, I-BET151) on ZSCAN4 mRNA levels insubconfluent FSHD1 myoblasts after 72 hours treatment. These dataemphasize the general nature of BET inhibition to reduce ZSCAN4 mRNAlevels presumably through inhibition of the production of DUX4.

A complimentary set of experiments was conducted in undifferentiatedmyoblasts to determine if treatment options with BETi could have lastingeffects on DUX4 inhibition even after treatment ceased. TO furtherclarify the utility of BET inhibition, FSHD1 and FSHD2 (MB200) myoblastswere grown continuously in low dose (100 nM) (+)-JQ1. ZSCAN4 mRNA levelswere measured at the steady state level during growth in low dose(+)-JQ1 and during a time course after withdrawal of (+)-JQ1 from thecultures. FIG. 10A shows a graph of the levels of ZSCAN4 mRNA in FSHD1myoblasts. The levels of ZSCAN4 remained decreased >95% compared tocontrol FSHD1 myoblast for 144 hours (6 days). FIG. 10B shows a graph ofthe levels of ZSCAN4 mRNA in FSHD2 myoblasts. The levels of ZSCAN4remained decreased >95% compared to control FSHD1 myoblast for 72 hours(3 days) and were still decreased by 75% at 96 hours after withdrawal of(+)-JQ1 from the cultures. These data indicate an extendedpharmacodynamic effect of BET inhibition that perhaps relates to theslow stochastic nature of the induction of DUX4 expression. Thissuggests that BETi may have a favorable therapeutic profile in thattheir pharmacodynamic effects (decreased DUX4 and downstream targetslike ZSCAN4) are extended relative to their pharmacokinetic profile(physical presence of drug).

(+)-JQ1 is generally accepted to inhibit the activity of all fourmammalian BET bromodomain proteins (BRD2, BRD3, BRD4 and BRDt).Therefore, inhibition of at least one of the four mammalian BETbromodomain proteins would appear to have a positive effect on FSHD. Inan embodiment, general inhibition of BET can be a useful treatment forFSHD and any BETi can be used. However, it is known that broad spectrumBETi often produce undesirable immunosuppressive effects which can leadto secondary infection and their use can lead to complicating sideeffects such as diarrhea.

Because of the above concerns, while one embodiment of the presentinvention provides that a broad spectrum BETi can be used to treat theeffects of FSHD, it is desirable to locate alternatives that may havefewer side effects. In an embodiment, this can be accomplished throughthe use of a selective BETi which targets only a subset of the relevantBET, ideally only one or two. Further, it may alternatively oradditionally be desirable to be able to provide a BETi in a limited doseand then remove it so long as the DUX4 expression remains inhibited.Such a treatment plan can reduce general immunosuppressive effectsallowing a patient's system to recover between treatments. BETi havetraditionally always been used with continuous or near continuoustreatments (e.g. treatments twice a day, every day) and a treatment planinvolving a single treatment, and then a long waiting period aregenerally unknown. The above, may achieve a similar effect in reducingDUX4, while reducing undesirable side effects.

While it is believed that general inhibition is suitable in anembodiment, in an alternative embodiment a combination of RNAiknockdowns and forced expression can be used to determine the specificBET bromodomain family members whose inhibition results in blockinginduction of DUX4 and its targets to allow for more accurate targetingof compounds, should such compounds become available and reduction ofside effects. Initially, determining the expression of all four BETgenes at the mRNA and protein levels in both normal and FSHD myoblastsin undifferentiated cells and during the course of differentiation canbe accomplished. This information alone may provide sufficient detailfor further refinement. For example, if the expression of one of the BETproteins increasing with differentiation can correlate with DUX4de-repression. The expression of BRD2, BRD3 and BRD4 is considereduniversal while, interestingly, BRDt is considered testis-specific andtherefore also provides for a further therapeutic possibility. The factthat forced DUX4 expression in vitro, FSHD muscle cells in vitro andFSHD patient muscle biopsies are all associated with aberrant malegermline gene expression patterns is intriguing in light of the factthat BRDt is a testis-specific BET protein whose function is requiredfor male germline gene expression. This initially implies that a BETispecific to BRDt also may be a suitable therapeutic.

In order to determine which gene target of BETi (BRD2, BRD3, BRD4, orBRDt) are necessary for DUX4 expression, and are thus therapeutictargets in FSHD, experiments were performed to identify which of thefour possible genes are the most likely target(s) responsible for DUX4expression.

FSHD1 myoblasts were transfected with Silencer Select siRNAs (Ambion,Life Technologies) on Day 0. On day 3, samples were harvested forwestern analysis. On day 4 RNA was harvested for qRT-PCR analysis. RNAsamples were done in triplicate and protein samples are singlets. Asseen in FIGS. 11A, 11B, 11C, and 12, BRD2 RNA and protein were bothselectively depleted with the BRD2 siRNA, BRD3 RNA and protein wereselectively depleted using the BRD3 siRNA, and BRD4 RNA and protein wereselectively depleted by either of the two BRD4 siRNAs as compared to thevarious controls.

As can be seen in FIGS. 13A, 13B, and 13C, DUX4 targets ZSCAN4, MBD3L2and TRIM43 were all specifically decreased only with the BRD4 siRNAs(and at a much reduced amount BRD2 siRNAs). These FIGS. indicate thatdepletion of BRD4, but likely not BRD2 or BRD3, results in decreasedDUX4 target gene expression and that BRD4 is the likely target of BETinhibitors that results in BET inhibitor-mediated decreases in DUX4 geneexpression. However, the data also indicate that BRD2 or BRD3 inhibitionmay still provide some benefit. Still further, the fourth BET protein,BRDt, was not reliably detected in FSHD myoblasts due to very lowexpression and siRNA directed towards BRDt did not affect DUX4 targetgene expression (not shown).

This disclosure is currently limited by the availability of highlyspecific inhibitors for individual gene targets, however, one ofordinary sill in the art would understand that BETi developed in thefuture that are selective to BRD4 would likely provide highly useful inthe treatment of FSHD. While the compounds identified herein here haveexcellent selectivity profiles for BET bromodomains over all otherbromodomains, there are currently few compounds that can selectivelyinhibit each isoform. However, such a compound would be expected to beuseful in the systems and methods discussed herein when discovered.

Continuous exposure of FSHD myoblasts and myotubes in culture to BETiblocked expression of DUX4 and therefore resulted in decreasedexpression of DUX4 target genes. To determine the exposure-responserelationship between BETi and DUX4 expression (pharmacodynamics) invitro, experiments were performed in both undifferentiated FSHDmyoblasts and differentiated FSHD myotubes.

In undifferentiated proliferating FSHD myoblasts, preliminaryexperiments showed that upon continuous exposure to BETi, decreases inDUX4 target gene expression required 72 h to manifest (not shown). Todetermine if continuous exposure for the entire 72 h period wasrequired, FSHD1 myoblasts were treated with 500 nM I-BET762 for variouslengths of time. At the end of each treatment period, compound wasremoved, the cells rinsed once and then re-fed with fresh media. At theend of the 72 h time point, RNA was harvested for gene expressionanalysis. FIGS. 14A-14G show that a 24 hour pulse of BETi was aseffective as a full 72 hour exposure in decreasing DUX4 target geneexpression (ZSCAN4, TRIM43, MBD3L2). This is surprising in that the drugtreatment effect was sustained after removal of compound. Myoblastlineage genes (MYF5, MYOD1) and SMCHD1 (gene that is mutated in FSHD2)were minimally affected by drug treatment. To further explore how longthe effects of drug treatment were sustained, FSHD1 myoblasts weretreated with a range of I-BET762 concentration for 24 hour beforerinsing and re-feeding with fresh media and harvesting at various timesup to 8 days after initial treatment. FIGS. 15A-15C demonstrate that theeffects of BETi on DUX4 target gene expression are sustained greaterthan 7 days after removal of drug. This unanticipated result issurprising because BETi have not previously been demonstrated to havelong lasting pharmacodynamic effects. Instead, they have been doseddaily in published studies, indicating a requirement for chronicexposure to compounds for sustained effects in animal models. Thus, inan embodiment of the present invention, BETi can be supplied for a pulsewindow (generally of at least 24 hours although shorter windows may beused) and then withdrawn for a period of time longer than the pulsewindow. Based on the above, this could be 7 days or more.

The exposure-response relationship between BETi and DUX4/target geneexpression was extended to differentiating FSHD myotubes. FIGS. 16A and16B demonstrate that a 24 hour pulse of BETi (+)-JQ1 temporarilydecreased myosin heavy chain (MYH2, differentiation marker for myotubes)gene expression, but that MYH2 expression rebounded to normal levelswhen measured 2 days after withdrawal of drug. On the other hand, DUX4target gene expression (ZSCAN4) continued to decrease 2 days afterwithdrawing drug. This shows that the pharmacodynamic response of DUX4is sustained and distinct from that of the differentiation marker MYH2which is only temporarily decreased. This again highlights thesurprising finding that BET inhibition results in sustained decreases inDUX4 gene expression. This may be explained by the infrequent andstochastic nature of DUX4 expression, which is detected in only afraction of cells in culture at any given time (e.g. 1 in 1,000 cells).The results here suggest that a 24 hour long pulse of BET inhibitiondisrupts ongoing DUX4 expression that cannot resume after drugwithdrawal. New DUX4 expression is presumably governed by the infrequentinitiation that occurs due to mutations causing FSHD.

While pulse treatment with most BETi allows recovery of thelineage-specific MYH2 gene expression in myotubes, it is surprising thatthe BETi RVX-208 did not suppress MYH2 expression upon continuousexposure. FIGS. 17A and 17B demonstrate that 20 μM RVX-208 does notreduce MYH2 expression while significantly reducing DUX4 target geneexpression. RVX-208 is different than other known BETi in that itpreferentially binds the second of the two bromodomains present in BETproteins (BD2). The functional consequence is that fewer genes areaffected by RVX-208. We have shown by two methods (pulseexposure-recovery with inhibitors such as I-BET762 and (+)-JQ1 andcontinuous exposure to the unique pharmacophore RVX-208) that blockingDUX4 expression via BET inhibition can be achieved without significantunwanted effects on muscle cell differentiation.

While the invention has been disclosed in connection with certainpreferred embodiments, this should not be taken as a limitation to allof the provided details. Modifications and variations of the describedembodiments may be made without departing from the spirit and scope ofthe invention, and other embodiments should be understood to beencompassed in the present disclosure as would be understood by those ofordinary skill in the art.

1. A method of reducing muscular weakness from facioscapulohumeralmuscular dystrophy (FSHD), the method comprising: administering to apatient with FHSD a therapeutic quantity of a BET inhibitor (BETi)sufficient to reduce DUX4 expression in said patient.
 2. The method ofclaim 1, wherein said BETi is selected from the group consisting of:(+)-JQ1, PFI-1, I-BET-762, I-BET-151, RVX-208, and CPI-0610.
 3. Themethod of claim 1 wherein said BETi is a selective inhibitor of BRD2. 4.The method of claim 1 wherein said BETi is a selective inhibitor ofBRD4.
 5. The method of claim 1 wherein said BETi is a broad spectruminhibitor.
 6. The method of claim 1 wherein in said administering, saidBETi is provided in an initial dose and a second dose is not providedfor at least 24 hours after said initial dose is finished.
 7. The methodof claim 6 wherein said initial dose is provided continuously over 24hours.
 8. The method of claim 6 wherein in said administering, said BETiis provided in an initial dose and a second dose is not provided for atleast 48 hours after said initial dose is finished.
 9. The method ofclaim 6 wherein in said administering, said BETi is provided in aninitial dose and a second dose is not provided for at least 72 hoursafter said initial dose is finished.
 10. The method of claim 6 whereinin said administering, said BETi is provided in an initial dose and asecond dose is not provided for at least 96 hours after said initialdose is finished.
 11. The method of claim 1, wherein said patient ishuman.